Proteomic is a method of choice for biomarker research, without a priori, it establishes a directory of proteins that are expressed in a cell, tissue or an entire organism. This approach has been implemented for proteomic analysis of colon cancer cells and phosphoproteomic analysis of liver biopsies.The study of colon cancer cells transformed by the cytosine deaminase and treated with the prodrug 5-fluorocytosine was performed by two-dimensional electrophoresis. Image analysis allowed the quantification of 353 proteins, 14 isoforms are overexpressed and 4 under expressed during treatment with the prodrug. Among the proteins whose expression is affected by the treatment, HSP90 has a constant level of expression, but is identified in two isoforms that differ in their pI. Mass spectrometry identified phosphorylation of ser 254 which could contribute to tumour regression.After developed an HPLC- TiO2 method for the purification of phosphopeptides, a proteomic analysis of 24 biopsies from human hepatocellular carcinoma on non-fibrous liver (nfCHC) and normal tissue was performed with the iTRAQ technology. Peptides overexpressed in tumours correspond to HSPs, DNA / RNA binding proteins (histones, spliceosome), proteins form Phase 1 detoxification (carboxyesterase, epoxide hydrolase), the cytoskeletal proteins (actinin, tubulin), antioxidant proteins or enzymes (superoxide dismutase, thioredoxin). The under-expressed peptides belong to proteins of the urea cycle, the detoxification (alcohol dehydrogenase) metabolism of sugars, lipids and amino acids. In the TiO2 fraction, 19 phosphopeptides are significantly overexpressed and 15 phosphopeptides were significantly under-expressed. This phosphoproteome has demonstrated an overrepresentation of the (S/T)P pattern among overexpressed phosphopeptides, indicating activation of proline-directed kinases in nfCHC or inhibition of the corresponding phosphatases. These deregulated peptides/proteins are as potential biomarkers for hepatocellular carcinoma.
Phosphoproteomics in cancer biomarkers discovery : chromatographic development and applications